what are 3 common dyes used to stain microscope specimens

Chapter iii - Microscopy and staining

iii - one Utilise of the microscope

The microscope, as shown in Figure iii-i, is one of the most important instruments utilized by the microbiologist. In guild to study the morphological and staining characteristics of microorganisms such every bit bacteria, yeasts, molds, algae and protozoa, you must exist able to apply a microscope correctly.

Figure 3.ane. The light microscope. A modern light microscope. This is an example of the kind used in the teaching labs at the University of Wisconsin-Madison. The various parts of the microscope are labeled. Please accept the time to become familiar with their names.

The compound microscope used in microbiology is a precision instrument; its mechanical parts, such every bit the calibrated mechanical phase and the adjustment knobs, are hands damaged, and all lenses, particularly the oil immersion objective, are delicate and expensive. Handle the musical instrument with care and keep information technology clean.

The microscope is basically an optical system (for magnification) and an illumination system (to make the specimen visible). To aid understand the part of the various parts of the microscope, nosotros will follow a ray of light as information technology works its way through a microscope from the low-cal source, through the lenses, up to the eye. Figure 3-8 traces the path of calorie-free through the parts of the microscope

Effigy 3.8. The path of lite through a microscope. Mod microscopes are complex precision instruments. Light, originating in the light source (one), is focused by the condensor (two) onto the specimin (3). The low-cal then enters the objective lens (iv) and the image is magnified. Lite then passes through a series of glass prisms and mirrors, eventually entering the eyepiece (five) where is information technology further magnified, finally reacing the eye.

Outset let u.s.a. consider a chief feature of all microscopes, the calorie-free source. Proper illumination is essential for effective use of a microscope. A tungsten filament lamp ordinarily serves as the source of illumination. If reflected illumination is used, a split lamp provides a focused beam of light which is reflected upward through the condenser lenses by a mirror.

The light from the illuminating source is passed through the substage condenser. The condenser serves two purposes; it regulates the amount of light reaching the specimen and it focuses the low-cal coming from the low-cal source. As the magnification of the objective lens increases, more than light is needed. The iris diaphragm (located in the condenser), regulates the amount of light reaching the specimen. The condenser as well collects the broad packet of lite produced past the lite source and focuses it on the small area of the specimen that is nether ascertainment.

Low-cal then passes upwards through the slide and into the objective lens where the first magnification of the epitome takes identify. Magnification increases the credible size of an object. In the compound light microscope ii lenses, one near the stage chosen the objective lens and another in the eyepiece, overstate the sample. The magnifying power of an objective lens is engraved in the lens mount. Microscopes in most microbiology laboratories have three objective lenses: the low power objective lens (10X), the high-dry out objective lens (40X) and the oil-immersion objective lens (100X). The desired objective lens is rotated into working position past means of a revolving nosepiece.

On both sides of the base of the microscope are the course and fine aligning knobs, used to bring the prototype into focus. Rotation of these knobs will either move the specimen and the objectives closer or farther autonomously. The coarse adjustment moves the nosepiece in large increments and brings the specimen into guess focus. The fine adjustment moves the nosepiece more slowly for precise concluding focusing. In some microscopes, rotation of the fine and course adjustment knobs will move the stage instead of the nosepiece.

Magnification alone is non the only aim of a microscope. A given picture may be faithfully enlarged without showing any increase in item. The true mensurate of a microscope is its resolving power. The resolving power of the lens is its ability to reveal fine detail and to make pocket-sized objects clearly visible. Information technology is measured in terms of the smallest distance between two points or lines where they are visible as divide entities instead of ane blurred image. The resolving power of the objective lens, engraved on the lens, allows us to predict which objective lens should exist used for observing a given specimen. However, having skillful resolution in the microscope does not guarantee a visible image, the resolving power of the human eye is quite limited. Often further magnification is needed to obtain a good paradigm.

When the oil-immersion objective lens is in use, the difference between the light-bending ability (or refractive index of the medium holding the sample) and the objective lens becomes important. Because the refractive index of air is less than that of glass, light rays are bent or refracted as they pass from the microscope slide into the air, as shown in Figure iii-9. Many of these low-cal rays are refracted at so bang-up an angle that they completely miss the objective lens. This loss of light is so astringent that images are significantly degraded. Placing a driblet of immersion oil, which has a refractive index like to glass, between the slide and the objective lens decreases this refraction, and increases the amount of calorie-free passing from the specimen into the objective lens. This results in greater resolution and a clearer image.

Figure 3.9. Refraction of light at 100X. Light passing out of the slide, into the air, toward the objective lens is refracted, due to the different in refractive alphabetize between air and glass. While the bending cause by this difference is non important at 100X and 400X, at 1000X this refraction is problematic, causing blurring of the image and significant loss of calorie-free. Immersion oil has a refractive index very similar to that of glass. Placement of a drop of oil betwixt the objective lens and the slide prevents the bending of light rays and clarifies the image. The blue dashed line represents a potential calorie-free ray if immersion oil is non present. The ruby dashed line represents a calorie-free ray if immersion oil is present.

The prototype of the specimen continues on through a series of mirrors and/or prisms that bend it toward the eyepiece. A further magnification takes identify at the eyepiece producing what is called a virtual epitome. Total magnification is equal to the product of the eyepiece magnification and the objective magnification. Most oft eyepiece lenses magnify 10-fold resulting in total magnifications of 100, 400, or 1000X, depending upon which objective is in place. Many mod microscopes will also have focusable eyepieces to compensate for differences between individuals and even between individual's eyes. The adjustment of these is important and is described below.

---

3 - 2 Operating procedure

Below we depict detailed directions for the use of a microscope. This will give you an appreciation of their operation. These directions have been written as by and large equally possible, but information technology may be necessary for your instructor to make modifications for the verbal microscopes you lot are using. Light microscopes used in didactics laboratories are designed for ease of apply and with some practice should go automatic.

1. Raise the nosepiece using the course adjustment knob. This provides greater admission for positioning the slide on the stage.

2. Rotate the nosepiece so that the 10X objective lens is in operating position.

3. Open the iris diaphragm approximately half mode.

4. Turn on the in-base of operations illuminator by depressing the push-type switch.

5. Place a stained specimen slide on the stage and with the naked eye position the specimen directly above the center of the condenser.

6. Apply the thumbwheel below the eyepieces to conform the interpupillary distance between the two eyepieces. This is important to be able to view specimens with both eyes, maximizing the quality of the image and preventing fatigue from prolonged use of i eye.

7. Movement the microscope condenser by means of the condenser adjustment knob until the meridian of the condenser is virtually at the highest position. There should be enough room to slide a slice of paper between the stage and the condenser, just no more. This will focus the light onto the slide.

8. Rotate the coarse aligning knob in a clockwise management to bring the 10X objective closer to the slide. View through the eyepieces and, without agonizing the coarse adjustment setting, slowly rotate the fine aligning knob until the specimen is in the sharpest possible focus.

9. The left eyepiece tube is focusable to compensate for refraction differences of the eyes. The correct procedure is to bring the specimen into sharp focus looking though the right eyepiece only. Then focus for the left eye by turning the left eye tube collar fully counter-clockwise. Adjacent, while viewing the specimen with the left eye only turn the knurled collar clockwise until the specimen is in sharp focus. Practise non conform the fine adjustment knob during this procedure.

10. Remove an eyepiece to view the dorsum aperture of the objective lens. Close the condenser iris diaphragm, then re-open until the leaves of the diaphragm merely disappear from view. Replace the eyepiece and view the specimen. The iris diaphragm may be airtight slightly to heighten contrast, specially when viewing unstained specimens.

    Unstained specimens have just minimal contrast with their surrounding environments. As a result they can normally exist viewed more effectively by setting the diaphragm at or most minimum opening. Reducing the diaphragm setting increases definition, contrast, and depth of focus but introduces diffraction bug and sacrifices resolution. Play with the diaphragm setting and select the best compromise by trial and error.

11. Once the specimen is in sharp focus using the 10X objective lens, it is and so possible to rotate the nosepiece to the 40X objective lens without irresolute the position of the fibroid adjustment knob. Very little refocusing with the fine adjustment knob is required since nearly light microscope objective lenses are parfocal. Call up that the iris diaphragm setting must exist inverse to permit more low-cal to laissez passer though the sample as the magnification increases.

12. If the specimen is to be viewed using the 100X oil immersion lens, immersion oil must be applied to the slide.

13. Rotate the 40X objective lens slightly to the side and so that a driblet of immersion oil may be placed on the specimen without getting it on the 40X lens.

14. Place a drop of immersion oil in the eye of the circumvolve of light formed on the specimen slide.

15. Advisedly plough the nosepiece until the 100X objective lens snaps into place. The objective lens should be in the oil but must not touch the slide.

16. Increase the low-cal intensity as required and rotate the fine adjustment knob to obtain a sharp focus of the specimen. If necessary make further adjustments to obtain optimal illumination.

If the microscope is not parfocal, it volition be necessary to lower the objective lens every bit shut to the slide as possible without touching it. This is done only while looking at the lens and slide from the side of the microscope. Bring the specimen into view by slowly raising the objective lens with the fibroid adjustment knob. Next, focus with the fine adjustment knob and adjust the illumination as necessary. If this is not successful the outset time, repeat the entire process.

In many cases, a preparation needs to exist observed just under the oil immersion lens. In this case, first locate the specimen and center information technology in the field with the depression power objective lens. And so add together oil and rotate the oil immersion objective lens into position.

---

3 - iii Common Problems

Certain bug real or apparent, may be encountered while operating your microscope. Here is a trouble shooting guide to help you if you are having difficulty focusing a sample.

The sample tin be focused at 10X, but it is difficult to find or blurry at 40X.

This is oft caused by immersion oil on the 40X lens. Wipe the 40X lens with lens newspaper to remove the oil and refocus. This can be prevented by never viewing a specimen with the 40X objective after calculation immersion oil to a slide.

The sample tin be focused at 10X only when the 40X lens is rotated in place it contacts the slide.

In about cases this is caused by the slide being identify on the phase upside downwardly †with the smear facing the stage. Check your slide carefully to brand certain it is placed on the stage correctly.

The fine adjustment knob does non plow in the direction required for sharp focusing.

This indicates that it has been turned to the limits of its threads, either upwardly or downward, every bit the case may be. Screw information technology back to about one-half the thread distance (About four turns), utilize the coarse aligning to raise or lower the objective lens sufficiently to bring the specimen into view; then refocus with the fine adjustment.

What I am viewing does not look like bacteria.

Bank check to brand sure you are in the correct focal plane that you are focusing on the smear and not dust on the lenses. To verify this, move the slide while looking at it. Anything in the smear should motility in the field of view.

What I am viewing does not look similar bacteria. Part Ii

If you are in the right focal plane, at that place may be issues with smear preparation. Did you estrus fix too much? Was the amount of culture applied sufficient? Did you lot stain the slide correctly? Many credible microscope bug can be attributed to poor slide preparation.

---

3 - 4 Proper care of the microscope

The post-obit rules, cautions and maintenance hints will aid keep your microscope in skillful operating status.

1. Use both easily when carrying the microscope: one firmly grasping the arm of the microscope; the other beneath the base. Avoid jarring your microscope.

To keep the microscope and lens systems clean:

2. Never bear on the lenses. If the lenses go muddied, wipe them gently with lens tissue.

3. If blurred specks announced in the field of view this may exist due to lint or smears on the eyepiece. If the specks move while rotating the eyepiece, the dust is on the eyepiece and cleaning the outer lens of the eyepiece is in order. If the quality of the prototype is improved past changing objective lenses, clean the objective lens with lens newspaper.

4. Never leave a slide on the microscope when it is not in apply.

5. E'er remove oil from the oil-immersion objective lens afterwards its use. If by blow oil should get on either of the lower-power objective lenses, wipe it off immediately with lens tissue.

6. Keep the stage of the microscope make clean and dry out. If whatever liquids are spilled, dry out the phase with a piece of cheesecloth. If oil should go on the stage moisten a slice of cheesecloth with xylol and clean the stage, so wipe it dry.

7. When non in use, store your microscope in its cabinet. Put the low power objective lens into position at its lowest point above the stage. Be sure that the mechanical phase does non extend beyond the edge of the microscope stage. Wrap the electric cord effectually the base.

   To avoid breaking the microscope:

8. Never strength the adjustments. All adjustments should work freely and hands. If anything does not work correctly, do not attempt to fix information technology yourself, immediately notify your instructor.

9. Never allow an objective lens to jam into or even to touch the slide or embrace-slip.

10. Never focus downward with the coarse aligning while you are looking through the microscope. E'er incline your head to the side with eyes parallel to the slide and sentinel the objective every bit yous move it closer to the slide. This will prevent you lot from smashing the objective into the slide.

11. Never exchange the objective or eyepiece lenses of unlike microscopes, and never under any circumstances remove the front lenses from objective lenses.

12. Never attempt to bear two microscopes at 1 time

If yous follow these rules, you volition never have trouble with your microscope.

---

3 - 5 Staining microorganisms

Preliminary identification of bacteria is usually based upon their cell morphology and grouping and the manner in which they react to certain staining procedures. The purpose of this department is to demonstrate some mutual staining reactions used to categorize microorganisms.

Effigy three.ii. An unstained bacterial smear. Unstained leaner are mostly fabricated of h2o and are nearly transparent when viewed through a lite microscope (pictured on the left). Note that most of the microbes are not visible, but a dust spec in the heart of the field of view is visible. Stains cling to the positive and negative charges of bacteria, but do non bind as readily to the background of a slide. They therefore differentiate microbes from their surroundings. Stained leaner are shown at 40X and 100X in the centre and right panels.

Unstained bacteria are practically transparent when viewed using the light microscope and thus are hard to see as shown in Effigy 3-two. The evolution of dyes to stain microorganisms was a significant advance in microbiology. Stains serve several purposes:

• Stains differentiate microorganisms from their surrounding environment

• They let detailed observation of microbial structures at loftier magnification

• Certain staining protocols tin help to differentiate betwixt different types of microorganisms.

Most dyes consist of two functional chemical groups equally shown in Figure 3-3. The chromophore group, which give dyes their characteristic color; and the auxochrome group, containing an ionizable chemic structure, which helps to solubilize the dye and facilitates binding to dissimilar parts of microorganisms. Previously, dyes were classified as acidic or bones, depending upon whether the pigment was negatively or positively charged at neutral pH. More accurately, dyes tin can be referred to as anionic (-) or cationic (+) and this is the convention that will be used in this manual. Cationic dyes (crystal violet, methylene blue) will react with groups on leaner that have a negative charge. Anionic dyes (eosin, nigrosine) will react with groups that have a positive accuse. Since most leaner accept many positive and negative groups in their prison cell walls and other surfaces, they will react with both cationic and anionic dyes.

Figure 3.3. The structure of crystal violet. The auxochrome groups of crystal violet is the charged carbon in the center of the molecule. This is typically neutralized by a Cl^- ion. The chromophore grouping consists of the 3 benzene rings and the central carbon. These structures readily absorb light.

Staining protocols tin be divided into 3 basic types, uncomplicated, differential, and specialized. Simple stains react uniformly with all microorganisms and only distinguish the organisms from their environs. Differential stains discriminate between various leaner, depending upon the chemical or physical composition of the microorganism. The Gram stain is an instance of a differential stain. Specialized stains detect specific structures of cells such as flagella and endospores.

---

three - 6 Preparation of a Bacterial Smear for Staining

Before staining and observing a microbe under a microscope, a smear must be prepared. The goal of smear grooming is to place an appropriate concentration of cells on a slide and then cement them there then that they do not wash off during the subsequent staining procedure. Figure three-iv demonstrates smear training.

The best smears are fabricated from bacteria that accept grown on a solid surface such as an agar camber or plate. A bit of growth from a culture is mixed with distilled or tap h2o to course a slightly turbid solution and this is spread on a clean grease gratuitous slide. When staining goop cultures, a drop of goop is transferred directly to a slide, using no extra water. The procedure for making a smear is as follows:

1. If more than than one culture is to be examined using the same stain, it is possible to prepare up to 6 smears on the same slide. Before preparing the slide, divide it into the appropriate number of sections and clearly characterization each section on the underside of the slide.

2. If your culture has been grown on a agar slant or agar plate. Place a small drop of h2o on a clean, grease-free slide. Side by side, using a sterile loop or straight wire needle, transfer a bit of the growth to the drop of water and rub the needle around until the cloth is evenly emulsified. Spread the drop over a portion of the slide to make a sparse movie. The suspension should be only slightly turbid.

3. If you are using a broth culture, the broth civilization must have clearly visible turbidity. Transfer a loopful of culture from the broth onto a make clean grease costless slide. Spread the drop over a portion of the slide to brand a thin film.

4. Permit the film to air-dry. To get a skilful stain, it is important to let the smear dry completely. Excess water left on the slide volition boil during the fixing stage, causing about microbe nowadays to rupture. Rushing this step will effect in a poor concluding stain.

5. One time dry, "ready" the smear to the slide past passing the bottom of the slide through the tip of the burner flame several times for a one second. After rut fixing, touch the heated portion of the slide to your paw. It should be comfortably warm, but not burning hot.

6. Accept care not to under-ready (the smear will wash off) or over-rut (the cells will exist ruptured or distorted) the slide. The correct amount of rut fixing is learned by experience.

7. Allow the smear to cool and apply the stain.

---

three - 7 The Uncomplicated Stain

In a elementary stain, the smear is stained with a solution of a single dye which stains all cells the same colour. Differentiation of cell types or structures is not the objective of the simple stain. However, certain structures which are not stained by this method may exist easily seen, for example, endospores and lipid inclusions.

Simple stains are, well elementary. One makes a smear and the applies a single stain to the slide. Below is a procedure for a simple stain.

1. Ready and heat-gear up a smear of the organism to exist studied.

2. Cover the smear with the staining solution. If crystal violet or safranin is used, allow 1 infinitesimal for staining. The use of methylene blue requires 3-five minutes to achieve good staining.

3. Carefully wash off the dye with tap water and blot the slide dry out with blotting paper, an absorbent paper pad or a paper towel.

3 steps, now wasn't that easy?

The above movie demonstrates the simple stain.

Figure 3-10 shows a light micrograph of what a unproblematic stain should await similar.

Figure 3.10. The Uncomplicated Stain. A photomicrograph of a simple stain at 1000X magnification. Note that all cells, regardless of species or cell wall construction, stain the same color.

---

3 - 8 The Gram Stain

The Gram stain, performed properly, differentiates well-nigh all bacteria into 2 major groups. For example, one group, the gram-positive bacteria, include the causative agents of the diseases diphtheria, anthrax, tetanus, ruddy fever, and certain forms of pneumonia and tonsillitis. A second grouping, the gram-negative bacteria, includes organisms which cause typhoid fever, dysentery, gonorrhea and whooping cough. In Leaner the reaction to Gram stain reagents is explained by different cell wall structures. Gram-positive microbes have a much thicker jail cell wall, while that found in Gram-negative microbes is thinner. Microbes from the Archaea domain incorporate different cell wall structures than that seen in microbes normally constitute in the lab (Leaner domain). Withal, they will still have a species specific Gram stain reaction, fifty-fifty though the underlying macromolecular structures are unlike.

The Gram stain is one of the about useful differential stains in bacteriology, including diagnostic medical bacteriology. The differential staining effect correlates to differences in the prison cell wall construction of microorganisms (at least Bacteria, but non Archaea as mentioned above). In order to obtain reliable results information technology is of import to take the following precautions:

• The cultures to be stained should be young - incubated in goop or on a solid medium until growth is just visible (no more than 12 to eighteen hours old if possible). Old cultures of some gram-positive leaner will appear Gram negative. This is especially true for endospore-forming leaner, such as species from the genus Bacillus. In this class, many of the cultures will accept grown for more than than 2 days. For virtually bacteria this is not a problem, merely be aware that some cultures staining characteristics may alter!

• When feasible, the cultures to be stained should be grown on a carbohydrate-free medium. Many organisms produce substantial amounts of capsular or slime cloth in the presence of certain carbohydrates. This may interfere with decolorization, and certain Gram-negative organisms such as Klebsiella may appear as a mixture of pinkish and purple cells.

Gram stain procedure

Below is a procedure that works well in the instruction laboratories.

1. Cover the slide with crystal violet stain and wait one infinitesimal.

2. Afterward ane minute wash the stain off (gently!) with a minimum amount of tap water. Bleed off almost of the water and proceed to the side by side pace. Information technology may help to hold the slide vertically and touch a bottom corner to newspaper toweling or blotting paper.

3. Cover the slide with iodine solution for one minute. The iodine acts as a mordant (fixer) and volition form a complex with the crystal violet, fixing it into the cell.

4. Rinse briefly with tap water.

5. Tilt the slide lengthwise over the sink and apply the alcohol-acetone decolorizing solution (dropwise) such that the solution washes over the entire slide from ane end to the other. All smears on the slide are to be treated thoroughly and every bit in this procedure. Process the sample in this manner for almost two-v seconds and immediately rinse with tap water. This process will decolorize cells with a Gram negative type of cell wall but not those with a gram-positive blazon of cell wall, equally a general rule. Drain off most of the water and proceed.

6. Equally the decolorized gram-negative cells need to be stained in order to be visible, cover the slide with the safranin counterstain for xxx seconds to ane minute.

7. Rinse briefly and blot the slide dry. Record each culture equally Gram positive (regal cells) or Gram negative (pink cells).

The to a higher place video demonstrates the Gram stain procedure, while Figure 3-11 shows the results of a Gram stain for gram-positive and gram-negative negative bacteria.

Figure 3.11. The Gram Stain. A photomicrograph of gram-positive and gram-negative leaner. Note that Gram reaction is dependent upon jail cell wall structure. A) E. coli a common gram-negative rod establish in the colon. B) Staphylococcus epidermidis a gram-positive cocci plant on the skin. C) Bacillus cereus a gram-positive rod constitute in the soil.

---

3 - nine The Endospore Stain

Cells of Bacillus, Desulfotomaculum and Clostridium (and several other, bottom-known genera--see Bergey's Manual) may, as a response to nutrient limitations, develop endospores that possess remarkable resistance to heat, dryness, irradiation and many chemic agents. Each cell can produce only one endospore. It is therefore not a reproductive spore every bit seen for some organisms such as Streptomyces and about molds. The endospore is essentially a specialized jail cell, containing a full complement of DNA and many proteins, merely niggling water. This dehydration contributes to the spores resistance and makes information technology metabolically inert. The endospore develops in a characteristic position (for its species) in the vegetative cell. Somewhen the cell lyses, releasing a costless endospore.

Endospore Stain Process

Endospore stains require estrus to bulldoze the stain into the cells. For a endospore stain to exist successful, the temperature of the stain must be near boiling and the stain cannot dry out out. Most failed endospore stains occur because the stain was immune to completely evaporate during the procedure.

1. Place the oestrus-fixed slide over a steaming water bathroom and identify a piece of blotting paper over the surface area of the smear. The blotting paper should completely cover the smear, but should non stick out past the edges of the slide. If it sticks out over the edges stain will period over the edge of the slide by capillary action and make a mess.

2. Saturate the blotting newspaper with the five-half-dozen% solution of malachite light-green. Allow the steam to heat the slide for 5 minutes, and replenish the stain if it appears to be drying out.

3. Cool the slide to room temperature. Rinse thoroughly and carefully with tap water.

4. Apply safranin for one infinitesimal. Rinse thoroughly but briefly with tap water, absorb dry and examine. Mature endospores stain green whether gratuitous or in the vegetative cell. Vegetative cells stain pink to red.

The above video demonstrates the endospore stain

Figure 3-12 shows a photomicrograph of an endospore stain.

Figure 3.12. The Endospore Stain. A photomicrograph of an enodspore stain. Spores present in the picture stain green, while the vegetative cells stain ruby. A) Staphylococcus epdiermidis which does not form endospores. B) The endospore-forming rod, Bacillus cereus.

---

iii - eleven Practice staining

In the written report and identification of bacteria, the microscope is indispensable! The series of micro-scopic observations in this do is designed to illustrate how leaner may exist viewed individually in their basic form, the cell. The second and 3rd periods herein coincide with those of Experiment ane where organisms isolated by the student are examined microscopically (and could exist establish to be more interesting than those provided in this practice!).

Menses 1

Materials

Hay infusions and diverse other items from nature

Slide with smears of Bacillus cereus and Staphylococcus epidermidis

Simple stain.

1. Yous are provided with a microscope slide with two smears. Following the directions for microscopy and staining, heat-fix the slide, making sure the slide goes through the flame smear-side up.
2. Gloves are available for the staining procedure. Placing the slide on the staining rack in the sink, comprehend the slide with crystal violet for one minute. For a review, look at the directions for the unproblematic stain.
3. Carefully rinse off the dye with tap water and blot the slide dry with newspaper towel or blotting newspaper.
4. With both hands, obtain the low-cal microscope from the cabinet (corresponding to your desk number). This is the type of microscope which we will always use to observe stained smears.
5. Unless the instructor has other directions more directly applicative to the microscope yous are using, use the uncomplicated process described in the operating process. Refer to this procedure equally you study the cells in the two smears in the following steps.
6. Identify the slide on the stage such that it is oriented every bit illustrated above. Make sure the clips on the stage hold the slide securely.
7. Begin your observations with the Bacillus cereus smear. (Encounter figure beneath) When observing this organism with the oil-immersion objective, you lot will notice that the cells are relatively large and rod-shaped (bacilli) and are usually in chains. Tape your observations on the next folio.
8. Echo this procedure with the Staphylococcus epidermidis smear. Cells of this organism are spheres (cocci) which are usually arranged in clusters (staphylococci) and pairs.
9. When you lot are through, be sure the microscope is put away properly (i.e., all oil wiped off, 10X objective lens in place, phase centered). Information technology is recommended that you go on the slide. (To remove immersion oil from smears, place a few pieces of lens paper on the slide to blot the oil. Then, add several drops of xylol to the lens paper. Peel the paper, now soaked with xylol, off the slide. Xylol is flammable! Keep it away from flames!)

Effigy 3.xiii. Elementary stain. A elementary stain of S. epidermidis and B. cereus. S. epidermidis (A), B. cereus old (B), B. cereus young (C)

Wet Mount

1. For observation of living microorganisms, various samples including a hay infusion are bachelor. To study the microorganisms in the aqueous materials available, it is necessary to brand moisture mounts. The procedure is relatively unproblematic:
2. Using a capillary pipette or inoculating loop, pick up some of the cloth from around the surfaces of grass and leaves and from the bottom of the sample. Place a drib of suspended material on a make clean microscope slide.
3. With a toothpick, spread a very thin layer of vaseline over a small part of the palm of your paw. Take a clean coverslip (e'er held by the edges) and gently scrape all four edges along your palm, picking up a thin line of vaseline forth each border.
4. Place the coverslip straight onto the driblet on the slide in such a mode that some air bubbles are trapped. Identify a small, multilayered piece of newspaper towel over the coverslip and press downward. Discard the slice of paper towel into the disinfectant.
5. Examine the wet mountain with your light microscope or a phase Dissimilarity microscope set up by the teacher at a special station in the back or side of the lab.
6. Without removing the coverslip, discard the slide into the disinfectant container on the stage. (Refer to page viii for cleanup directions.)

If you haven't already, Figure ane-2 presents a movie of the types of life forms constitute in a hay infusion.

Flow 2

Materials

Bacterial cultures growing either in a liquid medium (Centre Infusion Goop) or on a slant of an all-purpose medium followed by suspension in saline:

Escherichia coli - young civilisation, incubated 12-15 hours

Bacillus cereus - young civilization, incubated 12-xv hours

Bacillus cereus - old civilization, incubated ii-three days

Figure three.14. Gram stains. Gram stains of demonstration species. Below are shown typical Gram stain reactions of two species. E. coli (A), B. cereus old (B), B. cereus young (C). The images are slightly larger than what would be visible in a light microscope to meliorate clarity.

1. On one clean glass slide, set smears of the three cultures. Go to smear prepapration if you need a refresher. Simply when the smears have dried completely should the slide be heat-fixed.
2. Perform the Gram stain procedure as described.
3. As with whatever stained smear, definitive observations are made with the 100X, oil-immersion objective. Refer to the microscope directions already given, remembering to focus the slide initially with the 10X objective, moving and then to the oil immersion objective.
4. Go on in mind that the immature cultures of B. cereus and Eastward. coli are your positive and negative control cultures, respectively, for the observation of probable gram-variability of the older B. cereus culture.
5. Using the figures beneath, record your observations in your notebook, noting the Gram reaction (positive if purple, negative if red) and the cellular shape. Is there whatsoever departure seen between the ii cultures of Bacillus cereus? Is gram-variability evident for the older culture? Recall from the introduction to Experiment 1 that we can refer to one-time and immature cultures merely should not do then for individual cells. (Remember to discard the tubes and slides properly)

Period iii

Materials

This experiment will be done in class.

Immature bacterial cultures growing on slants of Center Infusion Agar:

Staphylococcus epidermidis

Pseudomonas fluorescens

An unknown

Record the number of your unknown!

Figure 3.15. Typical reactions of example strains for exam. The classic Gram reactions for Staphylococcus epidermidis (A) and Pseudomonas fluorescens (B). From this, determine whether they are Gram (+) or Gram (-). Notation we do not show an unknown every bit this must be done in course. The images are slightly larger than what would exist visible in a light microscope to improve clarity.

1. On a make clean glass slide, prepare rut-fixed smears of the three cultures, noting that these cultures are growing on a solid medium. Therefore the cells must be dispersed in a drop of h2o when preparing the smears, as a smear is always a dried intermission of cells. Take care not to make the smears also thick! S. epidermidis and P. fluorescens are your positive and negative control cultures (respectively) for your unknown.
2. Perform the Gram stain process and note the Gram reaction and cellular shape. Tape your results. Fill up out and turn in your clarification of your unkonwn. Relieve your slide until your graded unknown is returned.

Period 4

Materials

For the capsule stain:

36-48 hour civilisation of Klebsiella pneumoniae growing on a camber of EMB Agar (a high-sugar medium)

Dropper bottle of filtered India ink

For the acid-fast stain:

3-day culture of Mycobacterium smegmatis growing on a slant of Trypticase Soy Agar plus 1% glycerol

18-24 hour civilization of Micrococcus luteus (the negative control civilisation) growing in Nutrient Broth

Dropper bottles of carbol fuchsin (freshly-fabricated), acid alcohol and methylene blue

Capsule stain

Figure 3.16. The capsule stain. A sheathing stain using India ink at 1000x magnification. The cells of Klebsiella pneumoniaeare surrounded by a night groundwork. The sheathing is the articulate area surrounding the cells. The photomicrographs is slightly enlarged for clarity.

sheathing stain

1. Using the culture of Klebsiella pneumoniae, Place 1 loopful of water on a slide and emulsify in it a fleck of growth from the slant or plate culture of the designated organism. Add together a drop of filtered India ink to the prison cell break. It oftentimes works out well to place the drop of India ink adjacent to the prison cell suspension on the glass slide.
2. Obtain a clean coverslip (no fingerprints, smudges, dirt, etc.) and rim information technology lightly with vaseline; the vaseline can exist gently scraped from a thin layer applied to the palm of the manus. Place a pocket-sized, multi-layered piece (virtually 1-two cm2) of paper towel over the coverslip and press downwards firmly; discard the newspaper towel into the disinfectant.
3. Using the regular light microscope, focus initially with the 10X objective, switching to the 45X objective and so - if needed - the 100X, oil-immersion objective. Adjust the light intensity as required with the iris diaphragm. The outline of the cell can be seen inside the area of the clear sheathing.
4. Alternatively, the phase microscope can be used. Heed the precautions regarding apply of this microscope. Excellent observations tin can be made with but the 40X objective lens (which takes no immersion oil).
5. When finished, without removing the coverslip, discard the slide directly into the disinfectant. Never discard capsule stains and other moisture mounts with the stained smears, equally viable cells are still nowadays and the slides must be disinfected!. Record your observations below.

Acid-fast stain

Effigy three.17. The acid fast stain. A photomicrograph of Mycobacterium smegmatis (pinkish) and Micrococcus luteus (bluish) at 1000x magnification. G. smegmatis is acid-fast, retaining the carbol fuchsin dye, thus appearing pink. M. luteus is not acid-fast, loses the carbol fuchsin during decolorizaiton, and is counter-stained with methylene blue.

acid fast stain.

1. Set up a mixed smear of two organisms as follows: Identify a drop of the Micrococcus luteus broth culture on a slide. Into this drib, add together cells from the Mycobacterium smegmatis culture. Disperse the cells as much as y'all tin can (the Mycobacterium cells tend to clump), and set up a smear about the size of a nickel. Permit it air-dry completely, and and so rut-fix information technology well, passing the slide through the flame an actress one or two times.
2. Perform the acid-fast procedure (page 148, observing the slide with the regular light microscope) and record your observations below.
3. As with all stained smears, discard the slide in the appropriate container.

---

3 - 12 Summary of Microscopy and Staining

Staining and viewing microbes under the microscope is often necessary in for their identification and classification. The identity of a microbe can help in determining the cause of a disease or the source of food spoilage. Microscopes as well have important roles in genetics, cell structure, biochemistry and many other scientific disiplines. Hopefully, this brusque introduction has helped y'all to empathise the visualization of microorganisms.

---

lehmannlaught.blogspot.com

Source: https://bioinfo.bact.wisc.edu/instr/book/displayarticlesinchapter?theme=printer&cid=39

Share this post